Purificação, caracterização química e atividade de proteases e inibidores de proteases durante a germinação e em eventos pré e pós-germinativos de sementes de Parkia multijuga

Abstract

During seed germination, storage proteins are accumulated and degraded to support the major metabolic events involved in development new cells and tissues. The mobilization of proteins occurs through the activity of proteases, which can be regulated through the presence of proteases inhibitors that prevent premature hydrolysis of the storage proteins. The objective of this research was to investigate the activity of proteases and protease inhibitors during germination and pre-and postgermination of seeds of Parkia multijuga and purify trypsin inhibitors. Thereby, the seeds after breaking dormancy, they were germinated in plastic pots containing vermiculite and packed in germination chambers at 25ºC. The collection stages were quiescent seeds (SQ), after 24 h of imbibition (EM), radicle protrusion (RA), expansion of cotyledon node (NO) and issue shoot (PA). Proteins were extracted into 0.15 M NaCl. The activities of serine proteinases and trypsin and chymotrypsin inhibitors were performed using BAPNA and azocasein as substrates. The activity of cysteine proteases and papain and bromelain inhibitors using BANA and azocasein, respectively. Trypsin inhibitors were purified by affinity chromatography usin trypsin- Sepharose 4B and ion exchange on HiTrap DEAE FF. The purification and the mobilization of proteins was performed by electrophoresis on SDS-PAGE and 2D gels. During seed germination of P. multijuga observed a significant decrease in the protein content after RA remaining until PA was confirmed by 2D gels, wich show proteins degradation in the range of 35 to 75 kDa in PA, while 9 kDa proteins were synthesized during EM. Regarding the inhibitory activity, serine protease inhibition showed high stability during all stages studied, of which trypsin inhibition was 20 times higher than chymotrypsin. Inhibition of cysteine proteases is more variable, observing for maximum inhibition of papain and bromelain in EM and NO, respectively. The activities of serine and cysteine proteases presented themselves to constant NO, with an increase in PA. The purification of trypsin inhibitors involved two chromatographic steps, which were similar to the profiles obtained in all stages studied, indicating the purification of the same molecule or isoinhibitors in different stages studied. From the purification of trypsin inhibitors (PmTI1 and PmTI2) SQ, these proteins are characterized by presenting against trypsin inhibitory specificity, high stability against variation of temperature and pH and amino acid sequence homology to the Bowman-type inhibitors Birk from other Fabaceae species. Thus, serine and cysteine proteases inhibitors could participate in the regulation of protease activity during germination and events pre and post-germination of seeds of P. multijuga, with the trypsin inhibitors Bowman-Birk type present throughout the process of formation of the seedling.